Journal articles on the topic 'Quorum Stimuli'

Background: Quorum sensing is a cell-to-cell communication, which is extensively observed in bacteria. This process allows the cell to detect, analyze, share and act upon various environmental stimuli based on cell density. The molecular aspect of this process is the secretion and detection of chemical signaling molecules called autoinducers (AIs), which act upon the gene expression. The quorum sensing signaling pathway is specifically observed only bulk population or in other words, the quorum sensing is effective only in high cell density. The quorum sensing circuit in the bacterial population is widely studied under the following heading; quorum sensing in Gram positive bacterium, Quorum sensing in Gram negative bacterium and the Quorum sensing with respect to Interkingdom communication. These models are studied using the widely studied models like Vibrio fischeri in Gram negative QS circuit, Staphylococcus aureus in Gram positive QS circuit and Vibrio harveyi. This review paper details the introduction of quorum sensing and their gene level explanation and how they effect on the virulence of a particular species of bacteria. This paper also throws light on the realization that the bacteria has the capable of performing coordinated activities that was so long contributed to the eukaryotic cell performance.

ABSTRACT Numerous gram-negative bacteria employ a cell-to-cell signaling mechanism, termed quorum sensing, for controlling gene expression in response to population density. Recently, this phenomenon has been discovered in Escherichia coli, and while pathogenicE. coli utilize quorum sensing to regulate pathogenesis (i.e., expression of virulence genes), the role of quorum sensing in nonpathogenic E. coli is less clear, and in particular, there is no information regarding the role of quorum sensing during the overexpression of recombinant proteins. The production of autoinducer AI-2, a signaling molecule employed by E. coli for intercellular communication, was studied in E. coli W3110 chemostat cultures using a Vibrio harveyi AI-2 reporter assay (M. G. Surrette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046–7050, 1998). Chemostat cultures enabled a study of AI-2 regulation through steady-state and transient responses to a variety of environmental stimuli. Results demonstrated that AI-2 levels increased with the steady-state culture growth rate. In addition, AI-2 increased following pulsed addition of glucose, Fe(III), NaCl, and dithiothreitol and decreased following aerobiosis, amino acid starvation, and isopropyl-β-d-thiogalactopyranoside-induced expression of human interleukin-2 (hIL-2). In general, the AI-2 responses to several perturbations were indicative of a shift in metabolic activity or state of the cells induced by the individual stress. Because of our interest in the expression of heterologous proteins in E. coli, the transcription of four quorum-regulated genes and 20 stress genes was mapped during the transient response to induced expression of hIL-2. Significant regulatory overlap was revealed among several stress and starvation genes and known quorum-sensing genes.

The entomopathogenic fungus Ophiocordyceps sinensis is one of the best known and most precious medicines and health food in China. The blastospores-hyphae (dimorphism) transition of this fungus in host hemolymph is critical for the virulence and the mummification of host larvae. To regulate this transition, the effects of inoculum density and fifteen chemicals including fungal nutrients, fungal metabolites, quorum-sensing molecules (QSMs) and insect hormones on the dimorphism in O. sinensis were investigated in vitro. The blastospores tended to exhibit budding growth when inoculated at 107 blastospores per mL, and hyphal growth at concentrations lower than 106 blastospores per mL. At 105 blastospores per mL, the percentage of hyphal formation decreased with the addition of filtered spent medium containing 107 blastospores per mL, indicating the quorum-sensing effect. Blastospores-hyphae transition in this fungus by fifteen chemicals was varied from no response to dimorphic reversion. The addition of N-acetylglucosamine at three concentrations significantly stimulated hyphal formation while inhibiting budding growth. For the first time, insect hormone 20-hydroxyecdysone was found to be involved in the hyphal formation in fungi. These results open new possibilities to regulate the dimorphism, which would be beneficial for the cultivation of the Chinese cordyceps.

ABSTRACT Vibrio cholerae is the causative agent of the severe diarrheal disease cholera. A number of environmental stimuli regulate virulence gene expression in V. cholerae, including quorum-sensing signals. At high cell densities, quorum sensing in V. cholerae invokes a series of signal transduction pathways in order to activate the expression of the master regulator HapR, which then represses the virulence regulon and biofilm-related genes and activates protease production. In this study, we identified a transcriptional regulator, VqmA (VCA1078), that activates hapR expression at low cell densities. Under in vitro inducing conditions, constitutive expression of VqmA represses the virulence regulon in a HapR-dependent manner. VqmA increases hapR transcription as measured by the activity of the hapR-lacZ reporter, and it increases HapR production as measured by Western blotting. Using a heterogenous luxCDABE cosmid, we found that VqmA stimulates quorum-sensing regulation at lower cell densities and that this stimulation bypasses the known LuxO-small-RNA regulatory circuits. Furthermore, we showed that VqmA regulates hapR transcription directly by binding to its promoter region and that expression of vqmA is cell density dependent and autoregulated. The physiological role of VqmA is also discussed.

: Quorum Sensing (QS) is a phenomenon in which bacterial cells communicate with each other with the help of several low molecular weight compounds. QS is largely dependent on population density, and it triggers when the concentration of quorum sensing molecules accumulate in the environment and crosses a particular threshold. Once a certain population density is achieved and the concentration of molecules crosses a threshold, the bacterial cells show a collective behavior in response to various chemical stimuli referred to as “auto-inducers”. The QS signaling is crucial for several phenotypic characteristics responsible for bacterial survival such as motility, virulence, and biofilm formation. Biofilm formation is also responsible for making bacterial cells resistant to antibiotics. : The human gut is home to trillions of bacterial cells collectively called “gut microbiota” or “gut microbes”. Gut microbes are a consortium of more than 15,000 bacterial species and play a very crucial role in several body functions such as metabolism, development and maturation of the immune system, and the synthesis of several essential vitamins. Due to its critical role in shaping human survival and its modulating impact on body metabolisms, the gut microbial community has been referred to as “the forgotten organ” by O`Hara et al. (2006) [1]. Several studies have demonstrated that chemical interaction between the members of bacterial cells in the gut is responsible for shaping the overall microbial community. : Recent advances in phytochemical research have generated a lot of interest in finding new, effective, and safer alternatives to modern chemical-based medicines. In the context of antimicrobial research various plant extracts have been identified with Quorum Sensing Inhibitory (QSI) activities among bacterial cells. This review focuses on the mechanism of quorum sensing and quorum sensing inhibitors isolated from natural sources.

Objective: The present study was conducted to evaluate the antimicrobial and anti-quorum sensing (QS) ability of nanoparticles synthesized usingfruit wastes.Methods: QS is a system of response and stimuli correlated to population density. Bacteria by QS secrete certain signaling molecules called autoinducers. These bacteria also have a receptor that can specifically detect the signaling molecule known as an inducer. Since QS governs numerous processes in bacteria including virulence, QS inhibition promises to be an ideal target for the development of novel therapeutics. In the present study, silver nanoparticles (AgNPs) synthesized from the ginger, lemon peel, cinnamon, corn silk, pomegranate peel, and orange peel exhibited anti-QS properties by inhibiting violacein production in Chromobacterium violaceum assayed using agar well diffusion method.Results: AgNPs were synthesized from various cost-effective fruit waste sources. These AgNPs exhibited significant antibacterial and anti-QS properties.Conclusion: Hence, such sources can be explored for developing the effective therapy for urinary tract infections.Keywords: Silver nanoparticles, Quorum sensing, Urinary Tract infection.

Dutch elm disease (DED) is caused by the dimorphic fungi Ophiostoma ulmi, Ophiostoma novo-ulmi, and Ophiostoma himal-ulmi. A cell population density-dependent phenomenon related to quorum sensing was previously shown to affect the reversible transition from yeast-like to mycelial growth in liquid shake cultures of O. novo-ulmi NRRL 6404. Since the response to external stimuli often varies among DED fungal strains, we evaluated the effect of inoculum size on 8 strains of the 3 species of DED agents by determining the proportion of yeast and mycelium produced at different spore inoculum concentrations in defined liquid shake medium. The results show that not all DED fungi strains respond similarly to inoculum size effect, since variations were observed among strains. It is thus possible that the different strains belonging to phylogenetically close species use different signalling molecules or molecular signalling pathways to regulate their growth mode via quorum-sensing mechanisms.

ABSTRACTQuorum sensing (QS) is widely used by bacteria to coordinate behavior in response to external stimuli. InVibrio cholerae, this process is important for environmental survival and pathogenesis, though, intriguingly, a large percentage of natural isolates are QS deficient. Here, we show that QS-deficient mutants can spread as social cheaters by ceasing production of extracellular proteases under conditions requiring their growth. We further show that mutants stimulate biofilm formation and are over-represented in biofilms compared to planktonic communities; on this basis, we suggest that QS-deficient mutants may have the side effect of enhancing environmental tolerance of natural populations due to the inherent resistance properties of biofilms. Interestingly, high frequencies of QS-deficient individuals did not impact production of QS signaling molecules despite mutants being unable to respond to these inducers, indicating that these variants actively cheat by false signaling under conditions requiring QS. Taken together, our results suggest that social cheating may drive QS deficiency emergence withinV. choleraenatural populations.

Sophorolipid biosurfactants are biodegradable, less toxic and FDA approved. The purified acidic form of sophorolipid is stimuli-responsive with self-assembling properties and used for solubilizing hydrophobic drugs. This study encapsulated curcumin (CU) with acidic sophorolipid (ASL) micelles and analysed using photophysical studies like UV-visible spectroscopy, photoluminescence (PL) spectroscopy and time-correlated single photon counting (TCSPC). TEM images have revealed ellipsoid micelles of approximately 100 nm size and were confirmed by dynamic light scattering. The bacterial fluorescence uptake studies showed the uptake of formed CUASL nanostructures into both Gram-positive and Gram-negative bacteria. They also showed quorum quenching activity against Pseudomonas aeruginosa . The results have demonstrated this system has potential theranostic applications.

ABSTRACTDuring fermentation of sugars, a number of bacterial species are able to switch from mixed acid production to acetoin and 2,3-butanediol production in order to avoid lethal acidification of their environment, although the regulation of this switch is only poorly understood. In this study, we report the identification of thebudABstructural operon, involved in acetoin production inSerratia plymuthicaRVH1, and its activation by a LysR-type regulator encoded bybudR, immediately upstream of this operon. In addition, the regulation ofbudRtranscription was elucidated and found to be subject to negative control by BudR itself and to positive control by external stimuli such asN-(3-oxohexanoyl)-l-homoserine lactone (OHHL) quorum sensing signaling molecules and acetate. Interestingly, however, we observed that induction ofbudRtranscription by OHHL or acetate did not require BudR, indicating the involvement of additional regulatory factors in relaying these environmental signals to thebudRpromoter.

ABSTRACT Vibrio cholerae secretes a Zn-dependent metalloprotease, hemagglutinin/protease (HA/protease), which is encoded by hapA and displays a broad range of potentially pathogenic activities. Production of HA/protease requires transcriptional activation by the quorum-sensing regulator HapR. In this study we demonstrate that transcription of hapA is growth phase dependent and specifically activated in the deceleration and stationary growth phases. Addition of glucose in these phases repressed hapA transcription by inducing V. cholerae to resume exponential growth, which in turn diminished the expression of a rpoS-lacZ transcriptional fusion. Contrary to a previous observation, we demonstrate that transcription of hapA requires the rpoS-encoded σs factor. The cyclic AMP (cAMP) receptor protein (CRP) strongly enhanced hapA transcription in the deceleration phase. Analysis of rpoS and hapR mRNA in isogenic CRP+ and CRP− strains suggested that CRP enhances the transcription of rpoS and hapR. Analysis of strains containing hapR-lacZ and hapA-lacZ fusions confirmed that hapA is transcribed in response to concurrent quorum-sensing and nutrient limitation stimuli. Mutations inactivating the stringent response regulator RelA and the HapR-controlled AphA regulator did not affect HA/protease expression. Electrophoretic mobility shift experiments showed that pure cAMP-CRP and HapR alone do not bind the hapA promoter. This result suggests that HapR activation of hapA differs from its interaction with the aphA promoter and could involve additional factors.

ABSTRACT Pseudomonas aeruginosa releases a spectrum of well-regulated virulence factors, controlled by intercellular communication (quorum sensing) and mediated through the production of small diffusible quorum-sensing signal molecules (QSSM). We hypothesize that QSSM may in fact serve a dual purpose, also allowing bacterial colonization via their intrinsic immune-modulatory capacity. One class of signal molecule, the N-acylhomoserine lactones, has pleiotropic effects on eukaryotic cells, particularly those involved in host immunity. In the present study, we have determined the comparative effects of two chemically distinct and endobronchially detectable QSSM, N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) and 2-heptyl-3-hydroxy-4 (1H)-quinolone or the Pseudomonas quinolone signal (PQS), on human leukocytes exposed to a series of stimuli designed to detect differential immunological activity in vitro. 3-Oxo-C12-HSL and PQS displayed differential effects on the release of interleukin-2 (IL-2) when human T cells were activated via the T-cell receptor and CD28 (a costimulatory molecule). 3-Oxo-C12-HSL inhibited cell proliferation and IL-2 release; PQS inhibited cell proliferation without affecting IL-2 release. Both molecules inhibited cell proliferation and the release of IL-2 following mitogen stimulation. Furthermore, in the presence of Escherichia coli lipopolysaccharide, 3-oxo-C12-HSL inhibited tumor necrosis factor alpha release from human monocytes, as reported previously (K. Tateda et al., Infect. Immun. 64:37-43, 1996), whereas PQS did not inhibit in this assay. These data highlight the presence of two differentially active immune modulatory QSSM from P. aeruginosa, which are detectable endobronchially and may be active at the host/pathogen interface during infection with P. aeruginosa, should the bronchial airway lymphoid tissues prove to be accessible to QSSM.

Chronic and recurrent bacterial infections are frequently associated with the formation of biofilms on biotic or abiotic materials that are composed of mono- or multi-species cultures of bacteria/fungi embedded in an extracellular matrix produced by the microorganisms. Biofilm formation is, among others, regulated by quorum sensing (QS) which is an interbacterial communication system usually composed of two-component systems (TCSs) of secreted autoinducer compounds that activate signal transduction pathways through interaction with their respective receptors. Embedded in the biofilms, the bacteria are protected from environmental stress stimuli, and they often show reduced responses to antibiotics, making it difficult to eradicate the bacterial infection. Besides reduced penetration of antibiotics through the intricate structure of the biofilms, the sessile biofilm-embedded bacteria show reduced metabolic activity making them intrinsically less sensitive to antibiotics. Moreover, they frequently express elevated levels of efflux pumps that extrude antibiotics, thereby reducing their intracellular levels. Some efflux pumps are involved in the secretion of QS compounds and biofilm-related materials, besides being important for removing toxic substances from the bacteria. Some efflux pump inhibitors (EPIs) have been shown to both prevent biofilm formation and sensitize the bacteria to antibiotics, suggesting a relationship between these processes. Additionally, QS inhibitors or quenchers may affect antibiotic susceptibility. Thus, targeting elements that regulate QS and biofilm formation might be a promising approach to combat antibiotic-resistant biofilm-related bacterial infections.

Microorganisms are present in nearly every niche on Earth and mainly do not exist solely but form communities of single or mixed species. Within such microbial populations and between the microbes and a eukaryotic host, various microbial interactions take place in an ever-changing environment. Those microbial interactions are crucial for a successful establishment and maintenance of a microbial population. The basic unit of interaction is the gene expression of each organism in this community in response to biotic or abiotic stimuli. Differential gene expression is responsible for producing exchangeable molecules involved in the interactions, ultimately leading to community behavior. Cooperative and competitive interactions within bacterial communities and between the associated bacteria and the host are the focus of this review, emphasizing microbial cell–cell communication (quorum sensing). Further, metagenomics is discussed as a helpful tool to analyze the complex genomic information of microbial communities and the functional role of different microbes within a community and to identify novel biomolecules for biotechnological applications.

The brain is a complex and dynamic system, consisting of interacting sets and the temporal evolution of these sets. Electroencephalogram (EEG) recordings of brain activity play a vital role to decode the cognitive process of human beings in learning research and application areas. In the real world, people react to stimuli differently, and the duration of brain activities varies between individuals. Therefore, the length of EEG recordings in trials gathered in the experiment is variable. However, current approaches either fix the length of EEG recordings in each trial which would lose information hidden in the data or use the sliding window which would consume large computation on overlapped parts of slices. In this paper, we propose TOO (Traverse Only Once), a new approach for processing variable-length EEG trial data. TOO is a convolutional quorum voting approach that breaks the fixed structure of the model through convolutional implementation of sliding windows and the replacement of the fully connected layer by the 1 × 1 convolutional layer. Each output cell generated from 1 × 1 convolutional layer corresponds to each slice created by a sliding time window, which reflects changes in cognitive states. Then, TOO employs quorum voting on output cells and determines the cognitive state representing the entire single trial. Our approach provides an adaptive model for trials of different lengths with traversing EEG data of each trial only once to recognize cognitive states. We design and implement a cognitive experiment and obtain EEG data. Using the data collecting from this experiment, we conducted an evaluation to compare TOO with a state-of-art sliding window end-to-end approach. The results show that TOO yields a good accuracy (83.58%) at the trial level with a much lower computation (11.16%). It also has the potential to be used in variable signal processing in other application areas.

Over the past decades, Enterobacter spp. have been identified as challenging and important pathogens. The emergence of multidrug-resistant Enterobacteria especially those that produce Klebsiella pneumoniae carbapenemase has been a very worrying health crisis. Although efforts have been made to unravel the complex mechanisms that contribute to the pathogenicity of different Enterobacter spp., there is very little information associated with AHL-type QS mechanism in Enterobacter spp. Signaling via N-acyl homoserine lactone (AHL) is the most common quorum sensing (QS) mechanism utilized by Proteobacteria. A typical AHL-based QS system involves two key players: a luxI gene homolog to synthesize AHLs and a luxR gene homolog, an AHL-dependent transcriptional regulator. These signaling molecules enable inter-species and intra-species interaction in response to external stimuli according to population density. In our recent study, we reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. Whole genome sequencing and in silico analysis revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easI/R, in strain L1. In a QS system, a LuxR transcriptional protein detects and responds to the concentration of a specific AHL controlling gene expression. In E. asburiae strain L1, EasR protein binds to its cognate AHLs, N-butanoyl homoserine lactone (C4-HSL) and N–hexanoyl homoserine lactone (C6-HSL), modulating the expression of targeted genes. In this current work, we have cloned the 693 bp luxR homolog of strain L1 for further characterization. The functionality and specificity of EasR protein in response to different AHL signaling molecules to activate gene transcription were tested and validated with β-galactosidase assays. Higher β-galactosidase activities were detected for cells harboring EasR, indicating EasR is a functional transcriptional regulator. This is the first report documenting the cloning and characterization of transcriptional regulator, luxR homolog of E. asburiae.

It has long been known that phytopathogenic bacteria react to plant-specific stimuli or environmental factors. However, how bacterial cells sense these environmental cues remains incompletely studied. Recently, three kinds of histidine kinases (HKs) were identified as receptors to perceive plant-associated or quorum-sensing signals. Among these kinases, HK VgrS detects iron depletion by binding to ferric iron via an ExxE motif, RpfC binds diffusible signal factor (DSF) by its N-terminal peptide and activates its autokinase activity through relaxation of autoinhibition, and PcrK specifically senses plant hormone–cytokinin and elicits bacterial responses to oxidative stress. These HKs are critical sensors that regulate the virulence of a Gram-negative bacterium, Xanthomonas campestris pv. campestris . Research progress on the signal perception of phytopathogenic bacterial HKs suggests that inter-kingdom signalling between host plants and pathogens controls pathogenesis and can be used as a potential molecular target to protect plants from bacterial diseases. This article is part of the theme issue ‘Biotic signalling sheds light on smart pest management’.

Prokaryotic communities coordinate quorum behaviour in response to external stimuli to control fundamental processes including inter-bacterial communication. The obligate intracellular bacterial pathogen Chlamydia adopts two developmental forms, invasive elementary bodies (EBs) and replicative reticulate bodies (RBs), which reside within a specialised membrane-bound compartment within the host cell termed an inclusion. The mechanisms by which this bacterial community orchestrates different stages of development from within the inclusion in coordination with the host remain elusive. Both prokaryotic and eukaryotic kingdoms exploit ion-based electrical signalling for fast intercellular communication. Here we demonstrate that RBs specifically accumulate potassium (K+) ions, generating a gradient. Disruption of this gradient using ionophores or an ion-channel inhibitor stalls the Chlamydia lifecycle, inducing persistence. Using photobleaching approaches, we establish that the RB is the master regulator of this [K+] differential and observe a fast K+ exchange between RBs revealing a role for this ion in inter-bacterial communication. Finally, we demonstrate spatio-temporal regulation of bacterial membrane potential during RB to EB differentiation within the inclusion. Together, our data reveal that Chlamydia harnesses K+ to orchestrate host sensing, inter-bacteria communication and pathogen differentiation.

The major vault protein (MVP) mediates diverse cellular responses, including cancer cell resistance to chemotherapy and protection against inflammatory responses to Pseudomonas aeruginosa. Here, we report the use of photoactive probes to identify MVP as a target of the N-(3-oxo-dodecanoyl) homoserine lactone (C12), a quorum sensing signal of certain proteobacteria including P. aeruginosa. A treatment of normal and cancer cells with C12 or other N-acyl homoserine lactones (AHLs) results in rapid translocation of MVP into lipid raft (LR) membrane fractions. Like AHLs, inflammatory stimuli also induce LR-localization of MVP, but the C12 stimulation reprograms (functionalizes) bioactivity of the plasma membrane by recruiting death receptors, their apoptotic adaptors, and caspase-8 into LR. These functionalized membranes control AHL-induced signaling processes, in that MVP adjusts the protein kinase p38 pathway to attenuate programmed cell death. Since MVP is the structural core of large particles termed vaults, our findings suggest a mechanism in which MVP vaults act as sentinels that fine-tune inflammation-activated processes such as apoptotic signaling mediated by immunosurveillance cytokines including tumor necrosis factor-related apoptosis inducing ligand (TRAIL).

Quorum sensing (QS) is a cell-to-cell communication system that uses autoinducers as signaling molecules to enable inter-species and intra-species interactions in response to external stimuli according to the population density. QS allows bacteria such as Acinetobacter baumannii to react rapidly in response to environmental changes and hence, increase the chances of survival. A. baumannii is one of the causative agents in hospital-acquired infections and the number of cases has increased remarkably in the past decade. In this study, A. baumannii strain 863, a multidrug-resistant pathogen, was found to exhibit QS activity by producing N-acyl homoserine lactone. We identified the autoinducer synthase gene, which we named abaI, by performing whole genome sequencing analysis of A. baumannii strain 863. Using high resolution tandem triple quadrupole mass spectrometry, we reported that abaI of A. baumannii strain 863 produced 3-hydroxy-dodecanoyl-homoserine lactone. A gene deletion mutant was constructed, which confirmed the functionality of abaI. A growth defect was observed in the QS-deficient mutant strain. Transcriptome profiling was performed to determine the possible genes regulated by QS. Four groups of genes that showed differential expression were discovered, namely those involved in carbon source metabolism, energy production, stress response and the translation process.

ABSTRACT Rhizobium sp. strain NGR234 is a unique alphaproteobacterium (order Rhizobiales) that forms nitrogen-fixing nodules with more legumes than any other microsymbiont. We report here that the 3.93-Mbp chromosome (cNGR234) encodes most functions required for cellular growth. Few essential functions are encoded on the 2.43-Mbp megaplasmid (pNGR234b), and none are present on the second 0.54-Mbp symbiotic plasmid (pNGR234a). Among many striking features, the 6.9-Mbp genome encodes more different secretion systems than any other known rhizobia and probably most known bacteria. Altogether, 132 genes and proteins are linked to secretory processes. Secretion systems identified include general and export pathways, a twin arginine translocase secretion system, six type I transporter genes, one functional and one putative type III system, three type IV attachment systems, and two putative type IV conjugation pili. Type V and VI transporters were not identified, however. NGR234 also carries genes and regulatory networks linked to the metabolism of a wide range of aromatic and nonaromatic compounds. In this way, NGR234 can quickly adapt to changing environmental stimuli in soils, rhizospheres, and plants. Finally, NGR234 carries at least six loci linked to the quenching of quorum-sensing signals, as well as one gene (ngrI) that possibly encodes a novel type of autoinducer I molecule.

ABSTRACTVibrio choleraesenses its environment, including the surrounding bacterial community, using both the second messenger cyclic di-GMP (c-di-GMP) and quorum sensing (QS) to regulate biofilm formation and other bacterial behaviors. Cyclic di-GMP is synthesized by diguanylate cyclase (DGC) enzymes and degraded by phosphodiesterase (PDE) enzymes.V. choleraeencodes a complex network of 61 enzymes predicted to mediate changes to the levels of c-di-GMP in response to extracellular signals, and the transcription of many of these enzymes is influenced by QS. Because of the complexity of the c-di-GMP signaling system inV. cholerae, it is difficult to determine if modulation of intracellular c-di-GMP in response to different stimuli is driven primarily by changes in c-di-GMP synthesis or hydrolysis. Here, we describe a novel method, named theex vivolysate c-di-GMP assay (TELCA), that systematically measures total DGC and PDE cellular activity. We show thatV. choleraegrown in different environments exhibits significantly different intracellular levels of c-di-GMP, and we used TELCA to determine that these differences correspond to changes in both c-di-GMP synthesis and hydrolysis. Furthermore, we show that the increased concentration of c-di-GMP at low cell density is primarily due to increased DGC activity due to the DGC CdgA. Our findings highlight the idea that modulation of both total DGC and PDE activity alters the intracellular concentration of c-di-GMP, and we present a new method that is widely applicable to the systematic analysis of complex c-di-GMP signaling networks.

ABSTRACT Quorum sensing is a chemical communication process in which bacteria use the production, release, and detection of signal molecules called autoinducers to orchestrate collective behaviors. The human pathogen Vibrio cholerae requires quorum sensing to infect the small intestine. There, V. cholerae encounters the absence of oxygen and the presence of bile salts. We show that these two stimuli differentially affect quorum-sensing function and, in turn, V. cholerae pathogenicity. First, during anaerobic growth, V. cholerae does not produce the CAI-1 autoinducer, while it continues to produce the DPO autoinducer, suggesting that CAI-1 may encode information specific to the aerobic lifestyle of V. cholerae. Second, the quorum-sensing receptor-transcription factor called VqmA, which detects the DPO autoinducer, also detects the lack of oxygen and the presence of bile salts. Detection occurs via oxygen-, bile salt-, and redox-responsive disulfide bonds that alter VqmA DNA binding activity. We propose that VqmA serves as an information processing hub that integrates quorum-sensing information, redox status, the presence or absence of oxygen, and host cues. In response to the information acquired through this mechanism, V. cholerae appropriately modulates its virulence output. IMPORTANCE Quorum sensing (QS) is a process of chemical communication that bacteria use to orchestrate collective behaviors. QS communication relies on chemical signal molecules called autoinducers. QS regulates virulence in Vibrio cholerae, the causative agent of the disease cholera. Transit into the human small intestine, the site of cholera infection, exposes V. cholerae to the host environment. In this study, we show that the combination of two stimuli encountered in the small intestine, the absence of oxygen and the presence of host-produced bile salts, impinge on V. cholerae QS function and, in turn, pathogenicity. We suggest that possessing a QS system that is responsive to multiple environmental, host, and cell density cues enables V. cholerae to fine-tune its virulence capacity in the human intestine.

ABSTRACT Multihost bacteria have to rapidly adapt to drastic environmental changes, relying on a fine integration of multiple stimuli for an optimal genetic response. Erwinia carotovora spp. are phytopathogens that cause soft-rot disease. Strain Ecc15 in particular is a model for bacterial oral-route infection in Drosophila melanogaster as it harbors a unique gene, evf, that encodes the Erwinia virulence factor (Evf), which is a major determinant for infection of the D. melanogaster gut. However, the factors involved in the regulation of evf expression are poorly understood. We investigated whether evf could be controlled by quorum sensing as, in the Erwinia genus, quorum sensing regulates pectolytic enzymes, the major virulence factors needed to infect plants. Here, we show that transcription of evf is positively regulated by quorum sensing in Ecc15 via acyl-homoserine lactone (AHL) signal synthase ExpI and AHL receptors ExpR1 and ExpR2. We also show that the load of Ecc15 in the gut depends upon the quorum sensing-mediated regulation of evf. Furthermore, we demonstrate that larvae infected with Ecc15 suffer a developmental delay as a direct consequence of the regulation of evf via quorum sensing. Finally, we demonstrate that evf is coexpressed with plant cell wall-degrading enzymes (PCWDE) during plant infection in a quorum sensing-dependent manner. Overall, our results show that Ecc15 relies on quorum sensing to control production of both pectolytic enzymes and Evf. This regulation influences the interaction of Ecc15 with its two known hosts, indicating that quorum sensing signaling may impact bacterial dissemination via insect vectors that feed on rotting plants. IMPORTANCE Integration of genetic networks allows bacteria to rapidly adapt to changing environments. This is particularly important in bacteria that interact with multiple hosts. Erwinia carotovora is a plant pathogen that uses Drosophila melanogaster as a vector. To interact with these two hosts, Ecc15 uses different sets of virulence factors: plant cell wall-degrading enzymes to infect plants and the Erwinia virulence factor (evf) to infect Drosophila. Our work shows that, despite the virulence factors being specific for each host, both sets are coactivated by homoserine lactone quorum sensing and by the two-component GacS/A system in infected plants. This regulation is essential for Ecc15 loads in the gut of Drosophila and minimizes the developmental delay caused by the bacteria with respect to the insect vector. Our findings provide evidence that coactivation of the host-specific factors in the plant may function as a predictive mechanism to maximize the probability of transit of the bacteria between hosts.

Adenylate cyclases convert intra- and extracellular stimuli into a second messenger cAMP signal. Many bacterial and most eukaryotic ACs possess membrane anchors with six transmembrane spans. We replaced the anchor of the AC Rv1625c by the quorum-sensing receptor from Vibrio harveyi which has an identical 6TM design and obtained an active, membrane-anchored AC. We show that a canonical class III AC is ligand-regulated in vitro and in vivo. At 10 µM, the cholera-autoinducer CAI-1 stimulates activity 4.8-fold. A sequence based clustering of membrane domains of class III ACs and quorum-sensing receptors established six groups of potential structural and functional similarities. The data support the notion that 6TM AC membrane domains may operate as receptors which directly regulate AC activity as opposed and in addition to the indirect regulation by GPCRs in eukaryotic congeners. This adds a completely novel dimension of potential AC regulation in bacteria and vertebrates.

ABSTRACT Bacterial communities can sense their neighbors, regulating group behaviors in response to cell density and environmental changes. The diversity of signaling networks in a single species has been postulated to allow custom responses to different stimuli; however, little is known about how multiple signals are integrated and the implications of this integration in different ecological contexts. In the plant pathogen Pectobacterium wasabiae (formerly Erwinia carotovora), two signaling networks—the N-acyl homoserine lactone (AHL) quorum-sensing system and the Gac/Rsm signal transduction pathway—control the expression of secreted plant cell wall-degrading enzymes, its major virulence determinants. We show that the AHL system controls the Gac/Rsm system by affecting the expression of the regulatory RNA RsmB. This regulation is mediated by ExpR2, the quorum-sensing receptor that responds to the P. wasabiae cognate AHL but also to AHLs produced by other bacterial species. As a consequence, this level of regulation allows P. wasabiae to bypass the Gac-dependent regulation of RsmB in the presence of exogenous AHLs or AHL-producing bacteria. We provide in vivo evidence that this pivotal role of RsmB in signal transduction is important for the ability of P. wasabiae to induce virulence in response to other AHL-producing bacteria in multispecies plant lesions. Our results suggest that the signaling architecture in P. wasabiae was coopted to prime the bacteria to eavesdrop on other bacteria and quickly join the efforts of other species, which are already exploiting host resources. IMPORTANCE Quorum-sensing mechanisms enable bacteria to communicate through small signal molecules and coordinate group behaviors. Often, bacteria have various quorum-sensing receptors and integrate information with other signal transduction pathways, presumably allowing them to respond to different ecological contexts. The plant pathogen Pectobacterium wasabiae has two N-acyl homoserine lactone receptors with apparently the same regulatory functions. Our work revealed that the receptor with the broadest signal specificity is also responsible for establishing the link between the main signaling pathways regulating virulence in P. wasabiae. This link is essential to provide P. wasabiae with the ability to induce virulence earlier in response to higher densities of other bacterial species. We further present in vivo evidence that this novel regulatory link enables P. wasabiae to join related bacteria in the effort to degrade host tissue in multispecies plant lesions. Our work provides support for the hypothesis that interspecies interactions are among the major factors influencing the network architectures observed in bacterial quorum-sensing pathways. IMPORTANCE Quorum-sensing mechanisms enable bacteria to communicate through small signal molecules and coordinate group behaviors. Often, bacteria have various quorum-sensing receptors and integrate information with other signal transduction pathways, presumably allowing them to respond to different ecological contexts. The plant pathogen Pectobacterium wasabiae has two N-acyl homoserine lactone receptors with apparently the same regulatory functions. Our work revealed that the receptor with the broadest signal specificity is also responsible for establishing the link between the main signaling pathways regulating virulence in P. wasabiae. This link is essential to provide P. wasabiae with the ability to induce virulence earlier in response to higher densities of other bacterial species. We further present in vivo evidence that this novel regulatory link enables P. wasabiae to join related bacteria in the effort to degrade host tissue in multispecies plant lesions. Our work provides support for the hypothesis that interspecies interactions are among the major factors influencing the network architectures observed in bacterial quorum-sensing pathways.

ABSTRACTStaphylococcus aureusis a human pathogen causing a variety of diseases by versatile expression of a large set of virulence factors that most prominently features the cytotoxic and hemolytic pore-forming alpha-toxin. Expression of alpha-toxin is regulated by an intricate network of transcription factors. These include two-component systems sensing quorum and environmental signals as well as regulators reacting to the nutritional status of the pathogen. We previously identified the repressor of surface proteins (Rsp) as a virulence regulator. Acute cytotoxicity and hemolysis are strongly decreased inrspmutants, which are characterized by decreased transcription of toxin genes as well as loss of transcription of a 1,232-nucleotide (nt)-long noncoding RNA (ncRNA), SSR42. Here, we show that SSR42 is the effector of Rsp in transcription regulation of the alpha-toxin gene,hla. SSR42 transcription is enhanced after exposure ofS. aureusto subinhibitory concentrations of oxacillin which thus leads to an SSR42-dependent increase in hemolysis. Aside from Rsp, SSR42 transcription is under the control of additional global regulators, such as CodY, AgrA, CcpE, and σB, but is positioned upstream of the two-component system SaeRS in the regulatory cascade leading to alpha-toxin production. Thus, alpha-toxin expression depends on two long ncRNAs, SSR42 and RNAIII, which control production of the cytolytic toxin on the transcriptional and translational levels, respectively, with SSR42 as an important regulator of SaeRS-dependentS. aureustoxin production in response to environmental and metabolic signals.IMPORTANCEStaphylococcus aureusis a major cause of life-threatening infections. The bacterium expresses alpha-toxin, a hemolysin and cytotoxin responsible for many of the pathologies ofS. aureus. Alpha-toxin production is enhanced by subinhibitory concentrations of antibiotics. Here, we show that this process is dependent on the long noncoding RNA, SSR42. Further, SSR42 itself is regulated by several global regulators, thereby integrating environmental and nutritional signals that modulate hemolysis of the pathogen.

AbstractGene-expressing compartments assembled from simple, modular parts, are a versatile platform for creating minimal synthetic cells with life-like functions. By incorporating gene regulatory motifs into their encapsulated DNA templates, in situ gene expression and, thereby, synthetic cell function can be controlled according to specific stimuli. In this work, cell-free protein synthesis within synthetic cells was controlled using light by encoding genes of interest on light-activated DNA templates. Light-activated DNA contained a photocleavable blockade within the T7 promoter region that tightly repressed transcription until the blocking groups were removed with ultraviolet light. In this way, synthetic cells were activated remotely, in a spatiotemporally controlled manner. By applying this strategy to the expression of an acyl homoserine lactone synthase, BjaI, quorum-sensing-based communication between synthetic cells and bacteria was controlled with light. This work provides a framework for the remote-controlled production and delivery of small molecules from nonliving matter to living matter, with applications in biology and medicine.

AbstractCyclic di-GMP (c-di-GMP) transduces extracellular stimuli into intracellular responses, coordinating a plethora of important biological processes. Low levels of c-di-GMP are often associated with highly virulent behavior that depends on the type III secretion system (T3SS) effectors encoded, whereas elevated levels of c-di-GMP lead to the repression of T3SSs. However, extracellular signals that modulate c-di-GMP metabolism to control T3SSs and c-di-GMP effectors that relay environmental stimuli to changes in T3SS activity remain largely obscure. Here, we show that the quorum sensing signal autoinducer-2 (AI-2) induces c-di-GMP synthesis via a GAPES1 domain-containing diguanylate cyclase (DGC) YeaJ to repress T3SS-1 gene expression in Salmonella enterica serovar Typhimurium. YeaJ homologs capable of sensing AI-2 are present in many other species belonging to Enterobacterales. We also reveal that taurocholate and taurodeoxycholate bind to the sensory domain of the DGC YedQ to induce intracellular accumulation of c-di-GMP, thus repressing the expression of T3SS-1 genes. Further, we find that c-di-GMP negatively controls the function of T3SSs through binding to the widely conserved CesD/SycD/LcrH family of T3SS chaperones. Our results support a model in which bacteria sense changes in population density and host-derived cues to regulate c-di-GMP synthesis, thereby modulating the activity of T3SSs via a c-di-GMP-responsive T3SS chaperone.

Biofilm infections can be chronic, life threatening and challenging to eradicate. Understanding in vivo stimuli affecting the biofilm cycle is one step toward targeted prevention strategies. Iron restriction by the host is a stimulus for biofilm formation for some Staphylococcus aureus isolates; however, in some infection scenarios bacteria are exposed to abundant amounts of hemoglobin (Hb), which S. aureus is able to use as iron source. Thus, we hypothesized a role for Hb in the biofilm infection. Microplate “biofilm” assays showed biofilm-matrix production was increased in the presence of hemoglobin when compared to the provision of iron as an inorganic salt. Microscopic analysis of biofilms showed that the provision of iron as hemoglobin consistently caused thicker and more structured biofilms when compared to the effect of the inorganic iron source. Iron responsive biofilm gene expression analysis showed that Agr Quorum Sensing, a known biofilm dispersal marker, was repressed with hemoglobin but induced with an equivalent amount of inorganic iron in the laboratory strain Newman. The gene expression of two biofilm structuring agents, PSMα and PSMβ, differed in the response to the iron source provided and was not correlated to hemoglobin-structured biofilms. A comparison of the model pathogen S. aureus Newman with local clinical isolates demonstrated that while there was a similar phenotypic biofilm response to hemoglobin, there was substantial variation in the expression of key biofilm dispersal markers, suggesting an underappreciated variation in biofilm regulome among S. aureus isolates and that no general inferences can be made by studying the behavior of single strains.

ABSTRACT Bacteriophages (phages) have been proposed as alternative therapeutics for the treatment of multidrug-resistant bacterial infections. However, there are major gaps in our understanding of the molecular events in bacterial cells that control how bacteria respond to phage predation. Using the model organism Enterococcus faecalis, we used two distinct genomic approaches, namely, transposon library screening and RNA sequencing, to investigate the interaction of E. faecalis with a virulent phage. We discovered that a transcription factor encoding a LytR family response regulator controls the expression of enterococcal polysaccharide antigen (epa) genes that are involved in phage infection and bacterial fitness. In addition, we discovered that DNA mismatch repair mutants rapidly evolve phage adsorption deficiencies, underpinning a molecular basis for epa mutation during phage infection. Transcriptomic profiling of phage-infected E. faecalis revealed broad transcriptional changes influencing viral replication and progeny burst size. We also demonstrate that phage infection alters the expression of bacterial genes associated with intra- and interbacterial interactions, including genes involved in quorum sensing and polymicrobial competition. Together, our results suggest that phage predation has the potential to influence complex microbial behavior and may dictate how bacteria respond to external environmental stimuli. These responses could have collateral effects (positive or negative) on microbial communities, such as the host microbiota, during phage therapy. IMPORTANCE We lack fundamental understanding of how phage infection influences bacterial gene expression and, consequently, how bacterial responses to phage infection affect the assembly of polymicrobial communities. Using parallel genomic approaches, we have discovered novel transcriptional regulators and metabolic genes that influence phage infection. The integration of whole-genome transcriptomic profiling during phage infection has revealed the differential regulation of genes important for group behaviors and polymicrobial interactions. Our work suggests that therapeutic phages could more broadly influence bacterial community composition outside their intended host targets.

ABSTRACT Pseudomonas aeruginosa is an opportunistic human pathogen important to diseases such as cystic fibrosis. P. aeruginosa has multiple quorum-sensing (QS) systems, one of which utilizes the signaling molecule 2-heptyl-3-hydroxy-4-quinolone (Pseudomonas quinolone signal [PQS]). Here, we use hyperspectral Raman imaging to elucidate the spatiotemporal PQS distributions that determine how P. aeruginosa regulates surface colonization and its response to both metabolic stress and competition from other bacterial strains. These chemical imaging experiments illustrate the strong link between environmental challenges, such as metabolic stress caused by nutritional limitations or the presence of another bacterial species, and PQS signaling. Metabolic stress elicits a complex response in which limited nutrients induce the bacteria to produce PQS earlier, but the bacteria may also pause PQS production entirely if the nutrient concentration is too low. Separately, coculturing P. aeruginosa in the proximity of another bacterial species, or its culture supernatant, results in earlier production of PQS. However, these differences in PQS appearance are not observed for all alkyl quinolones (AQs) measured; the spatiotemporal response of 2-heptyl-4-hydroxyquinoline N-oxide (HQNO) is highly uniform for most conditions. These insights on the spatiotemporal distributions of quinolones provide additional perspective on the behavior of P. aeruginosa in response to different environmental cues. IMPORTANCE Alkyl quinolones (AQs), including Pseudomonas quinolone signal (PQS), made by the opportunistic pathogen Pseudomonas aeruginosa have been associated with both population density and stress. The regulation of AQ production is known to be complex, and the stimuli that modulate AQ responses are not fully clear. Here, we have used hyperspectral Raman chemical imaging to examine the temporal and spatial profiles of AQs exhibited by P. aeruginosa under several potentially stressful conditions. We found that metabolic stress, effected by carbon limitation, or competition stress, effected by proximity to other species, resulted in accelerated PQS production. This competition effect did not require cell-to-cell interaction, as evidenced by the fact that the addition of supernatants from either Escherichia coli or Staphylococcus aureus led to early appearance of PQS. Lastly, the fact that these modulations were observed for PQS but not for all AQs suggests a high level of complexity in AQ regulation that remains to be discerned.

The World Health Organisation has noted the ageing of the world’s population and has emphasised the growing need for older people’s needs to be considered in a range of contexts (WHO 1999, 2001). In Australia, people over the age of 65 currently constitute approximately 13% of the population (Australian Bureau of Statistics [ABS] 2002), but this number is expected to increase to 24% by 2051 (ABS 1999). As our population ages it will become increasingly necessary to cater to the particular needs of older audiences. This will involve a change in attitudes as the needs of this group have been largely neglected in western cultures and older people continue to be largely invisible in the media (Grossman 1998; Napoli 2002; Thomas and Wolfe 1995). The ubiquity of the written word and our reliance on this medium in almost every aspect of our lives signifies its importance in modern life. Reliance can become problematic when individuals experience difficulties in comprehending text-based communications (Akutsu, Legge, Ross, and Schuebel 1991). This article explains the problems older people can experience when attempting to read text and provides recommendations to enable communicators to enhance text comprehension among older audiences. Older audiences Older people tend to feel generally neglected by the media (Chafetz et al 1998). Previous studies have found that older people are not represented in line with their numbers (Szmigin and Carrigan 2001), and that negative stereotyping of older people is common in the media (de Luce 2001; Nielson and Curry 1997). This cultural bias is reflected in inadequate knowledge and accommodation of the special needs of older audiences when it comes to comprehending text. This has implications not just for the access of older people to sources of news and entertainment, but also for their ability to obtain information important to their health and well-being. Written materials such as brochures are frequently used to disseminate health-related information to older people (Clark et al 1999), and as such there is a need to ensure that we understand how to use text effectively for older audiences. Biological changes Quality of eyesight is closely correlated with age (Wahl and Heyl 2003). Deterioration in eyesight usually becomes noticeable in our 40s and 50s (Stuen and Faye 2003). Stuen and Faye (2003) have described the process by which the structure of the eye changes with age. They note that the lens of the eye thickens, hardens, and becomes yellowish in colour. Reduced elasticity in the lens and a tendency for the cornea to scatter light makes it more difficult for older people to focus their eyes, making reading problematic. The pupil shrinks with age, allowing smaller amounts of light to filter into the eye to assist with vision. This results in the need for brighter reading conditions, but not too bright as glare can also interfere with light entering the deteriorating cornea. As well as physical alterations to the eye, changes in cognitive capacity are also closely associated with ageing (Spotts and Schewe 1989). Wahl and Heyl (2003) postulate that the ageing of the central nervous system results in the deterioration of the neural pathways to the brain resulting in slower cognitive processing, including the processing of visual stimuli. Attentional capacity reduces with age, which means cognitive processing becomes more demanding (Moschis 1992). The outcome of these changes is that the older person experiences increasing difficulty in absorbing new information and evaluating unfamiliar stimuli (Moschis 1994). In particular, it appears more difficult for older audiences to remember new information that contradicts previously learned information (Rice and Okun 1994). Despite these changes in cognitive capacity, older people are reported as being able to assimilate information effectively if given ample time to do so (Moschis 1992; Tongren 1988). In addition, ensuring that new information relates to existing knowledge is likely to enhance comprehension and retention (Clark et al. 1999; Rice and Okun 1994). Implications for text style and presentation Age-related changes in visual acuity and cognitive processing result in the need for modifications in styles and presentation of text to maximise comprehension by older audiences. In terms of text style, font size may need to be slightly larger (Braus 1995), but not too large as people of all ages can experience reading difficulties when text is too large or too small (Akutsu et al. 1991). There are warnings against the use of all upper-case text as this impacts negatively on readability (Braus 1995). Colour may need to be manipulated to maximise contrasts to facilitate text discernment (Fairley et al 1997; Spotts and Schewe 1989). Black on white provides a high level of contrast, while shades of the same colour (for example, dark brown on light brown) provide much lower levels of contrast. Colours in the blue-green range can be particularly difficult for older eyes to discern (Braus 1995; Spotts and Schewe 1989). The use of bright colours such as red, orange, and yellow are recommended as they are relatively easy to distinguish (Clark et al. 1999). Text should be printed on matte rather than glossy paper and presented in non-glare environments to enhance readability (Braus 1995; Spotts and Schewe 1989). In terms of text presentation, the emphasis is on selecting an appropriate message and locating it carefully. There is general consensus that information should be confined to a small number of important points that are communicated simply and explicitly (Clark et al. 1999; Rice and Okun 1994; Spotts and Schewe 1989; Tooth, Clark, and McKenna 2000). This means using concrete terms whenever possible and using abstract terms only when necessary (Clark et al. 1999). It is important to ensure that extraneous information is excluded and the most pertinent information provided first to reduce processing workload (Spotts and Schewe 1989; Tooth et al. 2000). Repetition appears particularly critical in ensuring information is retained by older audiences (Clark et al. 1999). As older readers have greater trouble differentiating between previously learned information and new information (Clark et al. 1999), it is important to ensure that messages contain information that is related to existing knowledge in a way that will enhance assimilation (Rice and Okun 1994). It helps to locate information in uncluttered contexts and to use short lines and paragraphs (Fairley et al. 1997; Spotts and Schewe 1989). Using pictures to reinforce the message in the text can be effective (Moschis 1992), although the pictures should also be concrete rather than abstract (Clark et al. 1999). It is particularly important to test written materials designed for older audiences prior to dissemination to ensure the right messages are being received (Clark et al. 1999; Tooth et al. 2000). To conclude, there co-exists an awareness of population ageing and a cultural bias against older people that has resulted in relatively little knowledge of the optimal design of text for older people. This article has argued that the physical changes associated with aging have significant implications for the design and presentation of text. Steps should thus be taken to ensure that written communications are modified to better meet the needs of older audiences. Works Cited Akutsu, H., G. E. Legge, J. A. Ross, and K. J. Schuebel. "Psychophysics of Reading - X. Effects of Age-Related Changes in Vision". Journal of Gerontology 46.6 (1991): 325-331. Australian Bureau of Statistics. Older People, Australia: A Social Report, Canberra, 1999. Australian Bureau of Statistics. 2001 Census Basic Community Profile and Snapshot, Australia, Canberra, (2002. Braus, P. "Vision in an Aging America". American Demographics 17.6 (1995): 34-38 Chafetz, P. K., H. Holmes, K. Lande, E. Childress, and H. R. Glazer. "Older Adults and the News Media: Utilization, Opinions, and Preferred Reference Terms". Gerontologist 38.4 (1998): 481-489. Clark, K. L., R. AbuSabha, A. von Eye, and C. Achterberg. "Text and Graphics: Manipulating Nutrition Brochures to Maximize Recall". Health Education Research 14.4 (1999): 555-564. de Luce, J. "Silence at the Newsstands". Generations 25.3 (2001): 39-43. Fairley, S., G. P. Moschis, H. M. Meyers, and A. Thiesfeldt. "The Experts Sound Off". Brandweek 38.30 (1997): 24-25. Grossman, L. K. "Aging Viewers: The Best is Yet to Be". Columbia Journalism Review 36.5 (1998): 68. Moschis, G. P. Marketing to Older Consumers. Westport, Connecticut: Quorum, 1992. Moschis, G. P. Marketing Strategies for the Mature Market. Westport, Connecticut, Quorom, 1994. Napoli, P. N. "Audience Valuation and Minority Media: An Analysis of the Determinants of the Value of Radio Audiences". Journal of Broadcasting & Electronic Media 46.2 (2002): 169-184. Nielson, J. and K. Curry. "Creative Strategies for Connecting with Mature Individuals". Journal of Consumer Marketing 14.4 (1997): 310-322. Rice, G. E. and M. A. Okun. "Older Readers' Processing of Medical Information that Contradicts their Beliefs". Journal of Geronotology 49.3 (1994): 119-128. Szmigin, I. and M. Carrigan. "Learning to Love the Older Consumer". Journal of Consumer Behaviour 1.1 (2001): 22-34. Spotts, H. E. and C. D. Schewe. "Communicating with the Elderly Consumer: The Growing Health-Care Challenge". Journal of Health Care Marketing 9.3 (1989): 36-44. Stuen, C. and E. E. Faye. "Vision Loss: Normal and Not Normal Changes among Older Adults". Generations 27.1 (2003): 8-14. Thomas, V. and D. B. Wolfe. "Why Won't Television Grow Up?" American Demographics 17.5 (1995): 24 Tongren, H. N. "Determinant Behavior Characteristics of Older Consumers". Journal of Consumer Affairs 22.1 (1988): 136-157. Tooth, L., M. Clark, and K. McKenna. "Poor Functional Health Literacy: The Silent Disability for Older People". Australasian Journal on Ageing 19.1 (2000): 14-22. Wahl, H. and V. Heyl. "Connections Between Vision, Hearing, and Cognitive Function in Old Age". Generations 27.1 (2003): 39-45. World Health Organization. Action Towards Active Ageing. Geneva, (1999). Available: http://www.who.int/archives/whday/en/pages1999/whd99_8.html. Accessed 13/11/2001. World Health Organization. Health and Ageing: A Discussion Paper. Geneva, 2001. Citation reference for this article MLA Style Pettigrew, Simone. "Creating Text for Older Audiences" M/C: A Journal of Media and Culture <http://www.media-culture.org.au/0401/010-pettigrew.php>. APA Style Pettigrew, S. (2004, Jan 12). Creating Text for Older Audiences. M/C: A Journal of Media and Culture, 7, <http://www.media-culture.org.au/0401/010-pettigrew.php>